HPLC -problem with solvent peak - (Jul/20/2009 )

I guess no...it may affect the column. Look at the column manufacturers guidelines. Besides since it absorbs, you would have very high background and won't help in yr case.

Best,
TC

mimosa on Jul 23 2009, 09:00 PM said:

hexane can use as mobile phase for reverse phase column?
because i using c8 column

-T C-

i wouldn't use it alone but in combination with methanol and/or acetonitrile and/or water and/or chloroform or some other combination that allows you to separate your components without any of them coming out of solution.

-mdfenko-

mdfenko on Jul 23 2009, 03:59 AM said:

mimosa on Jul 22 2009, 09:01 AM said:

why the solvent peak in middle will affect the quantitation?
is that better the solvent peak elute at the beginning?

i try to dissolve the sample in methanol.
but the sample not totally dissolve because i could see the fat globule.

thank you

sorry, i thought you meant that the solvent peak came out with the other components (rising from the solvent peak). if the solvent peak is discrete then your quantitation will be unaffected (unless you are looking at percent of all peaks eluted, then i would turn off integration during the solvent elution).

what would worry me is that the sample is not completely solubilized by the mobile phase (methanol). i would expect the fat globules to form in the column as they separate from the hexane during the run.

can you run the analysis in hexane instead of methanol or, at least, add hexane to the mobile phase?

the sample will solubilized in fat or not?i running the fat soluble vitamin analysis.which solvent better used to dissolved besides hexane?
because the hexane will shorten the column life.

-mimosa-

mimosa on Jul 25 2009, 02:13 AM said:

the sample will solubilized in fat or not?i running the fat soluble vitamin analysis.which solvent better used to dissolved besides hexane?
because the hexane will shorten the column life.

we ran fat soluble vitamins on a c-18 column. i can't get my hands on the paper at the moment but the reference is:

it includes the extraction technique, solubilization, and the hplc conditions for fat soluble vitamins.

-mdfenko-

HPLC method for tocopherols I've found for my buddy uses C18 column and isocratic elution with mobile phase of 25:22:3 (v/v/v) methanol/acetonitrile/methylene chloride. After the extraction tocopherol is dissolved in hexane, then is exaporated to dryness and dissolved in mobile phase.

it includes the extraction technique, solubilization, and the hplc conditions for fat soluble vitamins.

according to u paper, the mobile phase that you use was ACN:MeOH:chloroform (47:47:6).
i extract the fat soluble vitamin by solid-liquid extraction. aceton:chroroform was used to extract the fat soluble vitamin
the problem here is the my sample contain allmost 65% fat.
will the fat affect the separation of fat soluble vitamin?
or better doing saponification to reduce the volume of fat?but the vitamink will degrade under alkaline condition.

according to u paper, the mobile phase that you use was ACN:MeOH:chloroform (47:47:6).
i extract the fat soluble vitamin by solid-liquid extraction. acetone:chloroform was used to extract the fat soluble vitamin
the problem here is the my sample contain almost 65% fat.
will the fat affect the separation of fat soluble vitamin?
or better doing saponification to reduce the volume of fat?but the vitamink will degrade under alkaline condition.

thank you

as long as the fat remains soluble it should separate on the column along with the vitamins.

we originally used this procedure (including sample preparation) with blood samples and for more than just vitamin e. fat in the sample did not seem to be a problem.

-mdfenko-

as long as the fat remains soluble it should separate on the column along with the vitamins.

we originally used this procedure (including sample preparation) with blood samples and for more than just vitamin e. fat in the sample did not seem to be a problem.

the problem of fat that it cannot dissolved in acetonitrile.
still could see the fat globular in acetonitrile.
do the fat globular entrapped those fat soluble vitamin?cause the fat soluble vitamin not dissolved in acetonitrile?
do SPE cartridge, c18 recommended use to clean up the sample?